HIV-1 infection is a world-wide epidemic. Although current drug therapies are effective at extending patient life, infections continue to spread, especially in non-developed countries, and the emergence of drug resistance has limited the effectiveness of many protease inhibitors. The mechanism by which the evolution of accessory mutations in HIV-1 protease (HIV-1PR) act to recover enzymatic function while imparting cross resistance to numerous inhibitors is the focus of this proposal. We are proposing a novel mechanism, which evokes protein conformational sampling, as a molecular basis to explain how secondary mutations accomplish this task. Specifically we hypothesize that mutations combine in patterns that alter HIV-1PR conformational sampling among four nominal states; namely, the closed, the semi- open, curled/tucked and wide-open such that (1) enzyme function is recovered by the secondary mutations when they combine to stabilize the semi-open conformation to a percentage observed in a native enzyme (e.g. inhibitor naive sequence) and (2) that cross resistance emerges when the mutations combine to stabilize open-like states such as the wide-open and curled/tucked conformations while concomitantly destabilizing the closed state population. It is through an innovative application of pulsed electron paramagnetic spectroscopy, which our lab has pioneered over the last five years that we measure conformational sampling ensembles in HIV-1PR. Within the aims of this proposal, we will make correlations among changes in HIV-1PR conformational sampling ensembles to enzymatic parameters, inhibition constants, inhibitor susceptibility, and viral fitness for numerous HIV-1PR variants. From these results, if our hypothesis is correct, we will provide a molecular level understanding of how secondary mutations elicit their effects on HIV-1PR. Additionally, these studies will also provide insights into explaining why the patterns of secondary mutation evolution against protease inhibitors are divergent in non-B subtypes. Finally, we will target numerous variants that show cross-resistance and increased conformational sampling of the open-like states for structure determination. Structures with more open-like conformations can serve as targets for the rational design of the new inhibitors for treatment of extremely resistant HIV-1PR variants.